Chromatography is generally known as a method for purifying biomolecules such as a protein. Specific examples include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography and affinity chromatography. An object protein can be highly purified by combining the processes. Therefore, the chromatography is an important technology in purification of erythropoietin, immunoglobulin or the like, to be used as a biomedicine, contained in blood, plasma or a culture liquid for a cell, for example. In particular, the affinity chromatography has a very higher selectivity to an object substance, as compared with other processes. Therefore, the object substance can be highly purified by a single operation, and the affinity chromatography is important as a technology by which a high productivity can be obtained in a short period of time of operation.
The affinity chromatography is performed by utilizing a ligand having an interaction with the object substance, and purification using the protein as the ligand may be performed. For example, an art is known in which an therapeutic antibodies is produced by purifying the immunoglobulins by using a protein A immobilized adsorbent, or a blood component such as fibronectin is purified by using a gelatin immobilized adsorbent.
The affinity chromatography using the protein as the ligand allows an efficient purification with a high selectivity. However, the affinity chromatography has a problem of degeneration or degradation of the ligand by alkaline washing, degradation of the ligand by protease, or deterioration of the ligand caused by weakness of the ligand per se.
Moreover, in the affinity chromatography using the protein as the ligand, an animal origin material may be mixed in a ligand material, and the affinity chromatography also has a problem of safety thereof. More specifically, a demand has been recently provided for avoiding use of a bovine origin gelatin as the ligand from a concern to zoonosis such as Bovine Spongiform Encephalopathy, and a ligand material in place of the animal origin material is required.
Furthermore, in the affinity chromatography using the protein as the ligand, conditions for recovering the object substance do not always become mild in consequence of a high affinity between the ligand and the object substance. For example, in one case, acidic conditions (pH 3, for example) are needed when allowing elution of the immunoglobulin from the protein A immobilized gel, and in another case, 8 M urea, 1 M NaBr, arginine or the like is used when allowing elution of the fibronectin from the gelatin immobilized gel (Non-patent literature No. 1). Under such severe elution conditions, the affinity chromatography has a fear of degeneration of the protein as the object substance, and expensiveness due to a high concentration of a reagent such as urea or arginine to be used for elution, and has a problem of cost and complicatedness thereof from necessity of treatment such as neutralization after an elution step.
Thus, an affinity ligand is required by which the protein can be purified in the purification of the protein under mild conditions, according to simple procedures, and at a low cost and safely while having a high affinity with the object substance and a high resistance to deterioration.
Meanwhile, a collagen well known as one of the biomolecules is a generic term for a protein having a triple helical structure formed of assembled three helices of polypeptides including repetitions of Gly-X-Y (wherein, X and Y represent various kinds of amino acids, X is Pro and Y is Hyp in many cases) in an identical direction.
A native collagen is known to be bonded with various kinds of biomolecules, or to have platelet aggregation capacity. A molecule such as a specific protein has been found to be bonded to a specific amino acid sequence in the native collagen so far (Non-patent literature No. 3). A study has been conducted for creating a collagen-like peptide to be bonded with a specific object molecule by utilizing such a finding. For example, a collagen-like peptide having a specific amino acid sequence to be bonded with collagen-binding molecules such as a von Willebrand factor is disclosed (Non-patent literature No. 2).
Moreover, a polypeptide having a repetition structure of a sequence of Pro-X-Gly (wherein X represents Pro or Hyp) as created as the collagen-like peptide has been found. The polypeptide has been found to have the platelet aggregation capacity (Non-patent literature No. 4), and a platelet aggregation induction substance containing the peptide has been disclosed (Patent literature No. 1). However, polypeptide fragments having the repetition structure of the sequence have not been known to be bonded with the fibronectin, the von Willebrand factor or the like with a high affinity.